Melnick- needles osteodysplasty presenting with quadriparesis.

Melnick-Needles syndrome or osteodysplasty, a monogenic heritable bone dysplasia, is characterized by a typical facies and characteristic radiological findings. Less than 70 well-documented cases have been reported in literature; most of them were sporadic. We report the first case from Eastern India in an adolescent male, who had cranio-vertebral junction anomalies and presented with spastic quadriparesis at the age of 13 years.

The role of calpain and calpastatin in the catabolism of erythrocyte-membrane proteins during anaemia in hamsters (Mesocricetus auretus) infected with Leishmania donovani.

The anaemia associated with visceral leishmaniasis is accompanied by altered Ca(2+) homeostasis and degradation of the cytoskeletal and integral proteins of the erythrocytic membrane. In the present study, such changes were followed in hamsters that were anaemic as the result of their experimental infection with Leishmania donovani. At each stage of the infection, the blood concentration of haemoglobin was found to be negatively correlated with the concentration of Ca(2+) (R(2) = 0.91), the percentage of erythrocytes with Heinz bodies (R(2) = 0.98) and thiol depletion (R(2) = 0.96) in the erythrocytes. Calpain (Ca(2+)-activated protease; EC 3.4.22.17) and its natural inhibitor calpastatin are known to regulate the catabolism of membrane structural proteins. Densitometric scanning of SDS-PAGE gels showed that erythrocytic membranes from infected hamsters contained less calpain and calpastatin than those from control animals. The level of calpain autolysis was found to increase as the infection progressed. The addition of purified calpain (from control hamsters) to erythrocyte ghosts caused greater degradation of the membranes of erythrocytes from infected animals than of the corresponding membranes from control animals. Calpastatin from the control hamsters was more effective, at inhibiting calpain-induced membrane proteolysis, than calpastatin from the infected animals. The results indicate that the Ca(2+)-activated protease and its inhibitor are involved in the degradation of erythrocytic membranes observed during visceral leishmaniasis.

Oxidative damage of erythrocytes: a possible mechanism for premature hemolysis in experimental visceral leishmaniasis in hamsters.

Visceral leishmaniasis is accompanied by severe anemia and pancytopenia. Reactive oxygen species are known to contribute to the pathogenesis of several red blood cell (RBCs) disorders. The present study reveals the extent of oxidative stress and the efficacy of the primary antioxidant system in erythrocytes of hamsters in the progressive anemic response at different stages of leishmanial infection. Increased intracellular precipitation of Heinz bodies secondary to oxidative denaturation of hemoglobin and enhanced formation of malonyldialdehyde suggest oxidative damage of erythrocytes, both in the hemoglobin and cell membrane, respectively. Decreased activities of superoxide dismutase and catalase in the infected animals indicate the generation of O2*- and H2O2, which in turn may produce the highly reactive *OH species. Decreases in the reduced glutathione level along with the decreased activities of glutathione reductase and glutathione peroxidase point to a deficient antioxidant defense system during the post-infection period. Accentuated degradation of both cytoskeletal and integral membrane proteins after 3 months of infection may eventually lead to membrane destabilization and early lysis of erythrocytes in experimental visceral leishmaniasis.

Altered calcium homeostasis and membrane destabilization in erythrocytes of hamsters infected with Leishmania donovani.

Homeostatic mechanisms regulating intracellular concentrations of Ca2+ at a low level are prerequisites for maintaining the integral and cytoskeletal structure of erythrocytes under normal physiological conditions. The present study was undertaken to assess the contribution of Ca2+ homeostasis in modifying red-cell stability in hamsters, during the anaemia caused by Leishmania donovani. Erythrocytes from the infected animals became increasingly fragile as infection progressed. This fragility may be the result of a gradual change in membrane permeability, as indicated by enhanced uptake of 45Ca2+. The increase in cytosolic Ca2+ and decrease in membrane-bound Ca2+ observed indicate the release of Ca2+ from the membrane store, leading to [Ca2+]i accumulation in the later stages of the post-infection period. Decline in the efficacy of Ca(2+)-effluxing enzyme may also contribute to the enhanced [Ca2+]i level, with subsequent degradation of membrane proteins in the erythrocytes of the infected animals. Marked inhibition of proteolytic degradation by the Ca(2+)-dependent thiol protease inhibitor leupeptin, with concomitant thiol depletion, indicates the involvement of Ca(2+)-induced thiol protease in the observed degradation of membrane proteins. The results indicate that an altered Ca2+ homeostasis in erythrocytes following leishmanial infection causes enhanced cellular accumulation of Ca2+, which in turn may lead to haemolysis in experimental visceral leishmaniasis.

Interaction of ascorbate and alpha-tocopherol enhances antioxidant reserve of erythrocytes during anemia in visceral leishmaniasis.

Visceral leishmaniasis (V.L.) is associated with enhanced lipid peroxidation along with impaired function of antioxidant defense system in erythrocytes. The effect of chronic treatment with ascorbate and alpha-tocopherol was studied on erythrocytes in hamsters infected with Leishmania donovani. Combination treatment with both antioxidants proved to be a potential suppressor of lipid hydroperoxide formation as well as hypotonic osmotic lysis during the leishmanial infection. Positive correlations between the depleted levels of erythrocyte ascorbate, GSH and alpha-tocopherol exhibit proportionate alterations in the nonenzymatic antioxidant levels at different stages of infection. Indirect measurement of transmembrane electron transfer as ferricyanide reduction suggests an active participation of endogenous contents of ascorbate and alpha-tocopherol in the protection against oxidative damage of membrane lipids. Cooperative behavior of both antioxidants in the ferricyanide reducing capacity was further evinced by resealing the ghosts in presence of exogenous ascorbate and alpha-tocopherol. Furthermore, intravesicular ascorbate serves in the defense of extravesicular ferricyanide induced oxidation of endogenous alpha-tocopherol. The results suggest an interacting role of ascorbate and alpha-tocopherol in maintaining the antioxidant reserve of erythrocytes during anemia in V.L.

Lipid peroxidation of erythrocytes in visceral leishmaniasis.

Lipid peroxidation of erythrocytes was studied in kala-azar patients having a considerable degree of anemia. Enhanced formation of oxidative metabolic products was observed in the erythrocytes of these patients. Decreased activities of the protective enzymes suggest impairment of the defense mechanism against peroxidative threat. These may contribute to some extent to the shortened lifespan of red cells in visceral leishmaniasis.

Elevated 2,3-diphosphoglycerate concentrations and alteration of structural integrity in the erythrocytes of Indian cases of visceral leishmaniasis.

The visceral leishmaniasis (VL) known as kala-azar in India is characterized by severe anaemia. The anaemia seems to be the result, at least in part, of the relatively short life-time of the erythrocytes, which have weakened cell membranes, possibly because of elevated concentrations of 2,3-diphosphoglycerate (2,3-DPG). There is a negative correlation (r = 0.91; P < 0.01) between erythrocytic 2,3-DPG concentrations and the blood concentration of haemoglobin, and the erythrocytes from infected patients display higher osmotic fragility than those from uninfected controls. Spectrofluorometry, using 1,6-diphenyl 1,3,5-hexatriene as a probe, indicated that fluorescence depolarization and microviscosity are also higher in the erythrocytic membranes from VL cases than in those from the controls. The cholesterol/phospholipid ratio is also relatively high in the membranes from the VL cases and there is degradation of the skeletal components and the major integral protein (band 3). The enhanced concentration of 2,3-DPG may be related to the altered structural integrity of the erythrocytes and this may lead to anisocytosis and the reduction in the erythrocytic half life.

Lipid peroxidation of erythrocytes during anemia of the hamsters infected with Leishmania donovani.

Visceral leishmaniasis has been found to be associated with severe anemia and premature lysis of erythrocytes. Peroxidative damage of red cells has been noted in several hemolytic anemias. Present study shows enhanced formation of methemoglobin in hamsters infected with Leishmania donovani. Increased formation of malonyldialdehyde and diene conjugate has been noted in the erythrocytes of the infected animals with the progress of anemia. Results showed decreased activities of protective enzymes like superoxide dismutase, catalase and glutathione reductase against peroxidative attack. An increase in the membrane cholesterol/phospholipid ratio and a decrease in membrane fluidity of erythrocytes were observed under the diseased condition. Densitometric scan after SDS-PAGE of red cell membrane of the infected animals revealed significant degradation of band 3 and band 4.1 proteins. The results suggest that alteration in the membrane may lead to reduced life span of the red cells in experimental visceral leishmaniasis.

Purification and partial characterization of a heparin-binding lectin from the marine clam Anadara granosa.

The heparin-binding lectin, Anadarin MS, from the plasma of the marine clam Anadara granosa is purified through affinity chromatography on heparin-Sepharose 4B followed by gel filtration on a Sepharose 6B column. The purified lectin is a pentameric protein of native M(r) 300 kDa and is composed of identical subunits of 60 kDa. The pI value of this Ca(2+)-dependent lectin is 6.2. Anadarin MS agglutinates normal rabbit erythrocytes but not that of human. Aspartic acid, glutamic acid, histidine and glycine are the predominant amino acids. Unlike other reported heparin-binding lectins, Anadarin MS exhibits a unique and strict specificity for iduronic acid containing glycosaminoglycans. This lectin agglutinates infective promastigotes of Leishmania donovani exclusively and can therefore be used as a novel biochemical surface marker for this parasite.

Purification and partial characterization of a N-glycolylneuraminic acid specific lectin from the clam Anadara granosa (L).

The N-glycolylneuraminic acid-specific lectin (AFL) from the foot muscles of the marine clam Anadara granosa has been purified to homogeneity by affinity chromatography on bovine submaxillary mucin-Sepharose 4B. The Ca(2+)-dependent lectin agglutinates rabbit erythrocytes. The purified lectin is a tetrameric protein of native M(r) 254 kDa having a pI value of 6.65. The M(r) of two subunits is 65 kDa each and that of the remaining two is 62 kDa each. The dominant amino acids of the lectin are aspartic acid, glutamic acid, serine and glycine. The lectin activity is inhibited only by N-glycolylneuraminic acid specially when it is present in the macromolecular structure of mucin viz., porcine submaxillary mucin, which is the most potent inhibitor. The binding site does not recognize N-acetylneuraminic acid. Due to this strict specificity, the lectin appears to be unique.

A novel galactosyl-binding lectin from the plasma of the blood clam, Anadara granosa (L) and a study of its combining site.

The marine blood clam species Anadara granosa (L) belong to arcidae, a family with some extraordinary haematological features. The plasma of this species exhibited strong haemagglutinating activities, from which a galactosyl binding lectin, Anadarin P, was purified in a single step affinity chromatography using Sepharose 4B-asialofetuin as an affinity matrix. The purified lectin, eluted with lactose, was found to be homogeneous by alkaline polyacrylamide disc gels, gel-filtration and isoelectric focusing. Native M(r) of the lectin was 130,000 having a PI value of 6.82 and was composed of two subunits of M(r) 17,000 and M(r) 16,000 which were noncovalently bound. The lectin was remarkably thermostable; the agglutinating titre remained unchanged over a wide range of pH (from 5 to 10) but increased with neuraminidase treated rabbit erythrocytes. Anadarin P combining site has been proposed to be small pocket-like structure which recognised only C-3 and C-4 hydroxyl groups of D-galactose. Presence of bulky groups at C-2 and C-6 exert strong steric hindrance as L-arabinose, 2-deoxy-D-galactose and D-xylose are better inhibitors than D-galactose. The lectin fails to differentiate methyl substituted galactosides as both alpha- and beta- methyl galactosides are equally active; but in case of substituted phenyl glycosides, the lectin shows different affinity towards alpha and beta anomers. The avidity of the lectin to bind the aromatic aglycons of galactosides suggests the presence of a hydrophobic region in the combining site. Interactions with some disaccharides indicate the presence of an extended area near the monosaccharide binding site.

Anemia in experimental visceral leishmaniasis in hamsters.

Experimental infection of hamsters with Leishmania donovani caused visceral leishmaniasis in which hematological changes occurred. The infected hamsters were anemic and reticulocyte counts were high. No significant change in the serum erythropoietin level was noted. Red cell membrane Na(+)-K(+)-ATPase and acetylcholinesterase activities increased. Osmotic fragility of the erythrocytes from infected animals increased. The level of 2,3-diphosphoglycerate of the red cells increased with the degree of anemia.

Effect of erythropoietin on the interaction of concanavalin A with rat erythrocytes.

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using 125I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with alpha-methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.

Effect of erythropoietin on membrane lipid peroxidation, superoxide dismutase, catalase, and glutathione peroxidase of rat RBC.

Starved animals having low levels of erythropoietin in blood showed increased MDA, fluorescent pigments, and met-Hb values whereas the hemoglobin concentration decreased significantly on starvation. In vivo and in vitro studies with Ep reversed the effects of starvation and brought these values close to normal. The activities of the enzymes (SOD, catalase, GSH-PX, GR G6PD, and 6PGD) which protect the RBC membrane directly or indirectly from peroxidative threat, decreased on starvation and restored to normal levels after Ep treatment.

Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells.

The effect of Ep on radioactive glucose and methyl-alpha-D-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [14C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-alpha-D-glucoside inhibited the Ep-stimulated glucose transport significantly. Ep also stimulated the transport of radioactive methyl-alpha-D-glucoside which was competitively inhibited in presence of D-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-alpha-D-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa.

Effect of erythropoietin on the different ATPases and acetylcholinesterase of rat RBC membrane.

The effect of Ep on different ATPases and acetylcholinesterase of rat RBC membrane was studied. Starvation caused a slight decrease in Mg2+-, Ca2+-, and Na+ + K+-ATPases. However, these enzyme activities were markedly increased on Ep treatment of starved rats. Specific activities of all three ATPases increased linearly with increasing concentration of Ep. Under identical conditions the hormone failed to stimulate the ATPase activity of liver plasma membrane. Desensitization by fluoride of allosteric inhibition of erythrocyte membrane-bound Na+ + K+-ATPase was observed under starvation which showed a return to normal n values on Ep administration. The enzyme from normal animals was inhibited almost completely at 0.1 mM fluoride whereas enzyme from starved and Ep-treated animals showed only about 50% inhibition at that fluoride concentration. Ep increased the acetylcholinesterase activity of normal RBC membrane to a small extent whereas the stimulation was much higher under starvation. The fluoride inhibition curve of this enzyme changed from sigmoidal to hyperbolic under starvation which again changed to allosteric on administration of Ep. These changes were closely correlated to n values. Red blood cells of Ep-treated animals became more susceptible to osmotic shock under the experimental conditions.

Effect of erythropoietin on the interchange of cholesterol and phospholipid between erythrocyte membrane and plasma.

Ep enhanced the exchange of unesterified cholesterol from plasma to the RBC membrane and vice versa. Similar to unesterified cholesterol, the exchange of phospholipids from plasma to the RBC membrane and vice versa in starved rats increased on the administration of Ep. But, unlike cholesterol exchange, the hormone favored phospholipid transport from the RBC membrane to plasma more significantly than from plasma to the RBC membrane.

Effect of erythropoietin on the lipid composition of red blood cell membrane.

Effect of Ep on [14C]acetate incorporation into different lipid fractions of RBC membranes in starved and phenylhydrazine-treated rats was studied. The incorporation was increased into both neutral and phospholipid fractions on Ep treatment to starved or phenylhydrazine-treated rats. A slight decrease in the ratio of neutral lipid to phospholipid was observed under the influence of Ep in starved rats (23%) or in phenylhydrazine-treated rats (36%). Incorporation of radioactivities into different phospholipid fractions of RBC membrane increased on Ep treatment to starved rats, whereas, the relative percentages of these phospholipids (except LPC) remained more or less unchanged under similar conditions. Phenylhydrazine treatment increased the relative percentage of PC and concomitantly decreased the percentage of Sph. Percentage composition of both these two phospholipids showed a tendency to return to their normal levels on administration of Ep to phenylhydrazine-treated rats. Ep decreased the sigma saturated/sigma unsaturated ratio of fatty acids in PE, PS, and PC of RBC membrane in starved rats. On the other hand, no significant change was observed in this ratio of fatty acids in the phospholipids except Sph of RBC membrane in the presence of phenylhydrazine and Ep. In Sph, the ratio went down under similar conditions.